As proteins age they are more likely to acquire molecular damage. To combat the functional decline of the proteome, most cellular proteins are rapidly turned over. In this way, potentially impaired polypeptides are constantly replaced with newly translated copies. Proteins with slower rates of turnover are therefore at greater risk of suffering a deleterious modification. Recently, long-lived proteins (LLPs) were discovered in post-mitotic cells of the rat central nervous system. Strikingly, these LLPs are associated with chromatin, the nuclear envelope, and the plasma membrane, all of which are cellular compartments that coordinate a myriad of regulatory functions. LLPs are therefore constantly exposed to potentially harmful metabolites. It is unknown how the functional integrity of these proteins is maintained over such a long time period, and whether the longevity of these proteins plays a specific role in long-lived cells. Thus, proposed experiments will test the specific hypothesis that LLPs regulate biological processes over extremely long time frames and that their functional decline drives cellular and organismal aging. This research proposal is designed to study the biology of LLPs, primarily focusing on nucleoporins and histones, and how they relate to post-mitotic tissue function. First, quantitative mass spectrometry and multi- isotope imaging mass spectrometry will be used to measure cell, organelle, and protein turnover rates in post-mitotic tissues in rats. Second, biochemical, cell biological, and genome-wide approaches will be used to determine the molecular properties of long-lived nucleoporins and histones. Efforts will be directed toward identifying and characterizing protein-repair pathways and protective post-translational modifications that are responsible for LLP longevity. In addition, it will be determined whether long-lived nucleoporins and histones serve as molecular timers to regulate nuclear pore function and gene expression, respectively. Finally, LLPs will be depleted in post-mitotic cells using a novel protein extraction and degradation system to determine how LLP impairment affects cellular function and aging. These analyses will focus on aberrant intracellular trafficking and gene regulation. Proposed studies promise to establish novel links between protein longevity and aging, and provide new molecular targets for understanding and potentially treating age-associated degenerative disorders.